The overall objective of the research proposed in this grant application is to continue our analysis the molecular pathways that regulate B lineage commitment and maturation. Our studies would be focused on the regulation and function of a subset of helix-loop-helix proteins, named E-proteins, in B-lineage development. E-proteins, E12, E47, HEB and E2-2, function at multiple stages thorughout B cell development. Specifically, E-proteins act: (1) to activate B-lineage specific gene expression, (2) to control Ig light chain gene rearrangement, (3) to regulate receptor editing, (4) to enforce the developmental checkpoint at the immature-B cell stage, (5) to[unreadable] regulate marginal zone versus follicular zone B cell development and (6) to induce AID gene expression. During the next grant cycle we would continue these studies. In particular we would investigate the relationship involving E2A, EBF and Pax-5 in early B lineage development. We would examine how E2A proteins activate and repress transcription and how they regulate Ig light chain gene rearrangement. We have recently demonstrated that the E2A[unreadable] proteins have the ability to repress transcription by recruitment of a transcriptional co-repressor, named ETO-2. We would examine the function of ETO-2 during B cell development and how the activity of ETO-2 is regulated. We have also recently determined using mass spectrometry two serine residues in E12 and E47 that are modified by phosphorylation and one lysine residue modified by methylation. We would introduce mutations in these residues and examine their impact on the activity of E2A both in vitro and in the mouse germ-line. We would further characterize the role of E-proteins in the regulation AID transcription. Overall the dissection of the roles of E2A in B cell development should help to clarify the mechanism by how E-proteins regulate B lineage maturation.[unreadable] [unreadable] AIMS[unreadable] Aim 1. To identify a hierarchical regulatory transcriptional network for specification to the B cell fate. We propose to examine how E12, E47, EBF and Pax5 act together to induce specification to the B cell fate.[unreadable] The various transcription factors would be expressed in E2A deficient hemaotopoietic progenitor cells. Transduced cells would be examined by microarray analysis for the expression of down-stream target[unreadable] genes. These data should provide a molecular framework by which progenitor cells are specified to become[unreadable] B-lineage cells.[unreadable] Aim 2. To examine how E2A proteins regulate transcription. Our data indicate that ETO-2 acts to repress E47 mediated transactivation. We would generate ETO-2 conditional deficient mice and would assess its role in lymphocyte development. We would examine at what stages of B cell development ETO-2 interacts with E47 to repress transcription. In vitro and in vivo analysis would be performed to determine how ETO-2 and E47 act together to regulate stage-specific B-lineage transcription arid Ig V(D)J gene rearrangement.[unreadable] Aim 3. To examine how E2A activity is regulated by post-translational modification. We have identified two phosphorylated serine residues located within or proximal to the E2A transactivation domains. One[unreadable] lysine residue is modified by methylation. We would mutate these residues and examine their role in mediating transcriptional repression and/or activation both in vitro and in the mouse germ-line. We would examine if and how phosphorylation and methylation of these residues is regulated during the various checkpoints in B cell development.[unreadable] Aim 4. To assess the mechanism by which E2A proteins regulate Ig K VJ recombination. We have recently established 3D-FISH technology to demonstrate looping of V regions within the IgH chain gene locus. We would analyze wild-type pro-B cells and pre-B cells for the presence of loops using 3D-FISH. B cells carrying mutation in the E2A binding sites present in the Ig light chain gene enhancer would be examined for looping. We would enforce E12 and E47 expression in E2A-deficient hematopoietic progenitor cells and examine for the presence of loops using 3D-FISH.[unreadable] Aim 5. To determine how AID transcription is regulated. During the previous grant cycle we showed that enforced expression of Id3 inhibits class switch recombination (CSR). To determine which of the Eproteins is required to activate AID expression and CSR we would analyze generate and analyze E12, E47, E2-2 and HEB deficient naive B cells for their ability to activate AID expression and CSR. To unambiguously determine whether the E-box sites present in the AID enhancer are indeed critical for function we would generate mice that carry target mutations in the binding elements. The mice would be analyzed for CSR and AID gene expression.[unreadable] [unreadable]